Our studies are concerned with various aspects of collagen metabolism. Prolyl and lysyl hydroxylases, enzymes which catalyze the hydroxylation of prolyl and lysyl residues in collagen during synthesis of this protein on polysomes bound to the membrane of the endoplasmic reticulum, require Fe ions, alpha-ketoglutarate, oxygen and a reducing compound. Ascorbic acid is the most efficient reducing compound but others such as tetrahydropterins, tetrahydrofolate, dithiothreitol and cysteine are also active to varying extents. The localization of these enzymes in microsomes as well as the specificity of the reducing compound are being studied. Since hydroxylation of prolyl residues in collagen is required for collagen secretion while hydroxylysine is required for the formation of crosslinks to form mature collagen fibers, the enzymes and their cofactors may play a role in the regulation of collagen synthesis and the maintenance of connective tissue. Collagen synthesis is also being used as a marker to study the effects of neoplastic transformation of a differentiated cell function. BIBLIOGRAPHIC REFERENCES: Evans, C., and Peterkofsky, B.: Ascorbate-independent proline hydroxylation resulting from viral transformation of BALB 3T3 cells and unaffected by dibutyryl cAMP treatment. J. Cell. Physiol. 89: 355-367, 1976. Peterkofsky, B. and Prather, W.: Cytotoxicity of ascorbate and other reducing agents towards cultured fibroblasts as a result of hydrogen peroxide formation. J. Cell. Physiol. 90: 61-70, 1977.